Nutrient supply systems and their effect on the performance of the Nile Tilapia (Oreochromis niloticus) and Lettuce (Lactuca sativa) plant integration system

The main aim of this work is to study the effect of different nutrient supply systems and their effect on the performance of the Nile Tilapia (Oreochromis niloticus) and Lettuce (Lactuca sativa var. crispa) plant integration system. To achieve that, five treatments having different culture systems (T1: Aquaculture (control), T2: Hydroponics (standard requirement: N = 210, P = 31, K = 234, Mg = 48, Ca = 200, S = 64, Fe = 14, Mn = 0.5, Zn = 0.05, B = 0.5, Cu = 0.02 and Mo = 0.01 ppm), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients (KNO3, 101 g L−1, KH2PO4, 136 g L−1, Ca(NO3)2, 236 g L−1, MgSO4, 246 g L−1, K2SO4, 115 g L−1 and chelates for trace elements) in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) were carried out. The previous systems were operated at three flow rates, namely, 1.0, 1.5 and 2.0 L h−1 plant−1. The various water quality parameters, plant growth and fish growth were studied. The result indicated that the highest values of N, P, k, Ca and Mg consumption rate were found with T2 and 1.5 L h−1 plant−1 of flow rate. The root length, fresh and dry of shoot and root for lettuce plants grown in T2 system was better than those grown in different culture system (T3, T4 and T5). Different culture systems showed significant effect on fish growth in terms of weight gain, specific growth rate and feed efficiency ratio. Higher growth rate was observed in treatment T3 as compared to other treatments. The production costs ranged from 2820.5 to 4885.4 LE ($ = 30.92 LE) for all culture systems.


Materials
The aquaponic system The system is a recirculate aquaponic system consists of fiberglass fish tanks, bio-sump tank, hydroponic units, pumps, air blower, water holding tank and reservoir, pipelines made of polyvinyl chloride (PE) were installed to connect the components of system (Fig. 1).
The fish tanks are cylindrical in shape and made of fiberglass with dimension of each tank are 1.0 m diameter and 1.0 m height.The capacity of each tank is 0.60 m 3 .The bio-sump tanks are made of fiberglass with dimension of 0.75 m diameter and 0.70 m height with a capacity of 0.25 m 3 , which are used in solid removal.These tanks have polyethylene sheets as a media to hold the bacteria (Nitrosomonas and Nitrobacter) from water.
Hydroponic unit include 12 tanks that made of polyethylene and rectangular in shape (dimensions of 80 × 40 × 30 cm for long, wide and high, respectively).The tanks were fixed on 1 m high from the ground with 2% slope and covered with foam board to fix the seedlings in it.Supplying irrigation water and solution to plants from water tank with the proper nutrient solution was carried out by 0.5 hp pump (Model First QB60-Head 25 m-Flow rate 30 L min −1 -Power 0.5 hp, China).The system has five circular polyethylene tanks with a 500 L capacity that used for collecting the drained solution by gravity from the ends of the systems.Air blower (Model C.C.P. Parma-Head 2.7 bar-Flow rate 10 m 3 h −1 -Power 1.0 kW, Italy) was used to supply the air to fish and hydroponic units, under various pressures through air stones.

Fish and plant species
• Nile Tilapia fish (Oreochromis niloticus) Tilapia nilotica (Oreochromis niloticus) fish has an individual weight of 60 g.Each tank got 70 fish at the beginning of work.Weight of fish was taken each 10 days.The flow rate was adjusted according to the growth rate based on the calculation of flow rate required equal fish weight divided carrying capacity (The fish carrying capacity of a water body is the maximum fish yield that can be carried by the natural bait organisms in the water body under the ideal natural conditions without feeding and fertilization).Carrying capacity is determined according to Khater 16 .Flow rate for fish ranged from 205 to 420 L min −1 .The daily feed rates at different fish sizes were carried out according to Rakocy 17 with feed pellet diameter according to Jauncey and Ross 18 .

• Lettuce
Lettuce seedlings were gown in the plastic cups (7 cm diameter and 7 cm height) filled with peat moss.The cups were irrigated daily using water with nutrient solution.Two weeks old lettuce seedlings were planted at 25.0 plant m −2 in the experimental tanks 19 .These seedlings were brought from the farm of Agriculture Faculty, Benha University.

Treatments
In this study, five treatments include (T1: Aquaculture (control), T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) and three flow rate for hydroponic units were 1.0, 1.5 and 2.0 L h −1 plant −1 .The experimental design was a split plot with four replicates.

Measurements
Water parameters.Water samples were taken at the influent and effluent of the system to measure pH, EC, temperature every day.Also, Total nitrogen (NH 3 , NO 2 and NO 3 ), Potassium, Phosphorus, Magnesium and Calcium were measured every 10 days at 10 am.EC Meter (Model ORION 230A-Range 0.0 to 19.99 ± 0.05, USA) was used to measured EC and temperature.pH Meter (Model ORION 105-Range 0.0 to 9999.9 ppm ± 0.5 ppm, USA) was used to measured pH.DO Meter (Model HANNA HI5421; Range: 0 to 90 mg L −1 ± 1.5%, Italy) was used to recorded dissolved oxygen.Spekol 11 (Model SPEKOL 11-Range 0. 1-1000 concentration ± 1 nm λ, UK) was used to measure ammonia, Nitrite and Phosphorus.Nitrate content was measured by using salicylic acid as described by Chapman 20 .Flame photometer (Model Jenway PFP7-Range 0. 1-999.9ppm ± 0.2 ppm, USA) was used to measured Potassium, Calcium and Magnesium.
The nutrient consumption rate was determined using Eq.(1) according to ASAE 21 : where NCR Nc is the nutrients consumption rate, mg day −1 plant −1 , NCR in is the nutrients at inlet of the hydroponic unit, mg L −1 , NCR out is the nutrients at outlet of the hydroponic unit, mg L −1 , Q is the discharge, L h −1 , n is the number of plants Plant parameters.Root length was measured every 10 days.The fresh and dry weight of shoot and root were measured at the end of the experiment.Dry weight the plants were measured by using oven dryer at 65 °C until constant weight was reached according to 22 .
Biological parameters of fish.Fish samples were taken every 10 days to determine the biological parameters which include: weight gain, specific growth rate, average daily gain, feed conversion ratio and feed efficiency ratio using the following equations: (1) where WG is the weight gained, g; W f is the mean final fish weight, g; W i is the mean initial fish mass, g; ADG is the average daily gain, g day −1 ; SGR is the specific growth rate, % day −1 or g day −1 ; t is the time, day, FCR is the feed conversion ratio, g feed g −1 fish; FER is the feed Efficiency ratio, g fish g −1 feed; FI is the feed intake, g; n f is the final number of fish in the tank.

Cost calculation for the different systems
The costs were calculated according to Khater 23 .Table 1 shows the input parameters of calculate total production costs of tilapia fish and lettuce plant for different culture systems.
Experiments and protocols were approved by the Research Committee in the Benha University.This study was carried out in compliance with the ARRIVE guidelines.This work is approved by the ethic committee at Benha University.All animal methods were carried out in accordance with relevant guidelines and regulations of Benha University.All the plant experiments and protocols were performed with relevant institutional, national, and international guidelines and legislation.

Statistical analysis
The data were subjected to analysis using statistical package SPSS version 21 in which two ways ANOVA and Duncan Multiple Range Test were conducted at 5% significant levels and 95% confidence limit to know the significant differences between the means of different parameters.

Water quality parameters
The water quality parameters for different culture systems (T1: Aquaculture (control), T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) during the growth period as shown in Table 2. Water temperatures are in the range of 25.37 ± 0.52 to 27.10 ± 0.36 °C for all culture systems under investigation.These systems have very close values of temperatures.Water temperature was within the range of 25.37 ± 0.52 to 27.10 ± 0.36 °C which is suitable for tilapia fish culture.Regarding the water pH, it varied within a range of 6.30 ± 0.23 to 6.91 ± 1.55, with no noticeable variations among the treatments.Different culture systems did not show any significant different on water pH which was found in desirable limits for fish as well as plant growth.Dissolved oxygen (DO) in water during the growth period varied within a range of 6.05 ± 0.89 to 6.51 ± 1.07 mg L −1 without significant variations during the growth period for the values of culture systems.The DO values fluctuated and varied nevertheless there were within narrow range and they were found within optimum limits for the fish as well as plant culture.Electrical conductivity (EC) values of water in The input parameters of calculate total production costs of tilapia fish and lettuce plant for different culture systems.

Cost item Units
Culture system T1 T2 T3 T4 T5 Regarding ammonia-N generation rate, the results reveal that ammonia values were higher in T1 and T3 system than other systems.Ammonia-N generation rate depends on the feeding rate in the system which was adjusted after each sampling as per the fish body weight.The lowest Nitrite-N concentration was observed in T3 (0.16 ± 0.03 mg L −1 ) as compared to T1, T4 and T5.Nitrate-N concentration (NO 3 -N) in T2 and T4 which were significantly higher than that of T1, T3 and T5 systems.Nitrate-N is relatively less toxic to fish and is not a health hazard except at exceedingly high levels above 300 mg L −124 .The phosphorus concentration values were 10.64 ± 2.06, 27.33 ± 2.38, 6.17 ± 1.55, 25.79 ± 2.50 and 7.39 ± 1.39 mg L −1 for T1, T2, T3, T4 and T5 culture systems, respectively.Phosphorus concentration was found significantly higher in the T2 and T4 system as compared to other systems.Potassium, calcium and magnesium concentrations also showed similar patterns.

Nutrients consumption
Table 3 shows the nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) consumption rate by lettuce plants during the growth period in different culture systems (T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) and different flow rates (1.0, 1.5 and 2.0 L h −1 plant −1 ).At 1.0 L h −   13.00 mg plant −1 were found with T3 and 2.0 L h −1 plant −1 of flow rate.These results agreed with those obtained by 25,26 whose found that the highest values of nutrients consumption rate of plant were found with a flow rate of 1.5 L h −1 plant −1 .
The statistical analysis showed that the differences between the obtained data of nutrients consumption rate of lettuce plants due to the effect of culture system (A) and flow rate (B) were significant.The analysis showed also that the interaction between both AB was significant.

Root length
The root length of lettuce plants grown in different culture systems (T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) and different flow rates for hydroponic units (1.0, 1.5 and 2.0 L h −1 plant −1 ) during the experimental period are shown in Fig. 2. It could be seen that the results indicate that the root of the lettuce plant grown in different culture system increases with increasing flow rate and plant age.It was noticed that there was not any overlapping (interference) between roots of the growing plants as a result of choosing a suitable distance (20 cm) apart between plants during different growth stages.If there is www.nature.com/scientificreports/any overlapping existed it was very limited (not more than 5.0%).These results were in agreement with 16 found that the plant spacing for lettuce was (20-25 cm).
The results also indicate that the root length for lettuce plants grown in T2 system was taller than those grown in different culture system (T3, T4 and T5).It could be seen that the highest value of root length was 25.32 ± 2.55 cm was found with T2 and 2.0 L h −1 plant −1 of flow rate, while, the lowest value of root length was 19.67 ± 1.86 cm was found with T3 and 1.0 L h −1 plant −1 of flow rate.Generally, the growth of plant in the hydroponic system depend on the optimum conditions and the amounts of nutrients available to root system and the oxygen available, which has relationship with the temperature and pressure.These results are in agreement with those obtained by 27 .Regarding the effect of flow rate, the results indicate that the root length increases with increasing flow rate.It which ranged from 3.28 ± 0.39 to 25.32 ± 2.55 cm depending on all treatments under study.
Multiple regression analysis was carried out to obtain a relationship between the root length of lettuce plants as dependent variable and different both of culture system, flow rate (1.0, 1.5 and 2.0 L h −1 ) and experimental period (1 to 50 day) as independent variables.The best fit for this relationship is presented in the following equation: where RL is the root length of lettuce plant, cm, PA is the lettuce plant age, day, Q is the flow rate, L h −1 .

Fresh and dry weight of shoot
Fresh and dry weight of shoot of lettuce plants grown in different culture systems (T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) and different flow rates (1.0, 1.5 and 2.0 L h −1 plant −1 ) at the end of growth period (50 days) are shown in Fig. 3a,b.The results indicate that the fresh and dry weight of shoot of lettuce plants grown in T2 system were better than those of different culture system (T3, T4 and T5).It could be observed that the fresh weight of shoot of lettuce plants were 329.40 ± 12.91, 387.23 ± 13.08 and 350.48 ± 15.37, 250.84 ± 9.92, 273.38 ± 11.12 and 261.00 ± 9.86, 296.01 ± 15.54, 325.56 ± 10.01 and 303.92 ± 13.33 and 303.60 ± 8.95, 359.09 ± 10.13 and 338.47 ± 14.07 g plant −1 for 1.0, 1.5 and 2.0 L h −1 plant −1 , respectively, for T2, T3, T4 and T5 systems at the end of growth period.While, the dry weight of shoot of lettuce plants were 40.02 ± 1.57, 43.17 ± 2.09 and 41.99 ± 2.11, 32.01 ± 0.99, 35.33 ± 1.44 and 33.96 ± 1.72, 33.97 ± 1.60, 40.95 ± 1.27 and 37.11 ± 3.15 and 36.46 ± 0.98, 41.72 ± 2.13 and 39.05 ± 3.00 g plant −1 for 1.0, 1.5 and 2.0 L h −1 plant −1 , respectively, for T2, T3, T4 and T5 systems at the end of growth period.The variations in growth and yield of lettuce plants are explained by the variation of nutrients available and flow rates.Generally, the growth of lettuce is dependent on the nutrients available under proper conditions of temperature and pressure besides the balance of nutrients and oxygen supply.These results were in agreement with 28 .
The results indicate that the highest values of fresh and dry weight of shoot (387.23 ± 13.08 and 43.17 ± 2.09 g plant −1 ) were found with T2 and 1.5 L h −1 plant −1 of flow rate, while, the lowest values of fresh and dry weight of shoot (250.84 ± 9.92 and 32.01 ± 0.99 g plant −1 ) were found with T3 and 1.0 L h −1 plant −1 of flow rate.These results agreed with those obtained by 25,26,29 whose found that the highest values of fresh and dry weight of plant were found with a flow rate of 1.5 L h −1 plant −1 .

Fresh and dry weight of root
Fresh and dry weight of root of lettuce plants grown in different culture systems (T2: Hydroponics (standard requirement), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in (7)  For T2 RL 2 = −3.74+ 0.52PA + 1.88Q R 2 = 0.98 www.nature.com/scientificreports/water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) and different flow rates (1.0, 1.5 and 2.0 L h −1 plant −1 ) at the end of growth period (50 days) are shown in Fig. 4a,b.The results indicate that the fresh and dry weight of root of lettuce plants grown in T2 system were better than those of different culture system (T3, T4 and T5  and 9.89 ± 0.64 g plant −1 for 1.0, 1.5 and 2.0 L h −1 plant −1 , respectively, for T2, T3, T4 and T5 systems at the end of growth period.These findings may be referred to that the plants in hydroponic system (T2) are grown at precise control over the nutrient solution and ability of them to be in their most favorable growing condition.These results were in agreement with 30,31 .

Fish weight
Figure 5 shows the individual fish weight for different culture systems (T1: Aquaculture (control), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) during the experimental period.The results indicate that the individual fish weight in different culture system increases with increasing experimental period.It could be seen that the individual fish weight significantly increased from 60.00 ± 1.28 to 143.06 ± 2.09, 60.00 ± 1.28 to 144.08 ± 2.44, 60.00 ± 1.28 to 121.01 ± 1.97 and 60.00 ± 1.28 to 143.49 ± 3.51 g, when the growth period increased from 1 to 50 days, respectively, for T1, T3, T4 and T5 systems.The results also indicate that the highest value of individual fish weight (144.08 ± 2.44 g) was found for T3 culture system, while, the lowest value of individual fish weight (121.01 ± 1.97 g) was found for T4 culture system.
The results indicate that the highest values of the fish growth rate, specific growth rate and feed efficiency ratio were 1.68 ± 0.40 g day −1 , 1.75 ± 0.26% day −1 and 0.96 ± 0.05 g fish g −1 feed, respectively, were found for T3 culture system, while, the highest value of the feed conversion ratio was 1.58 ± 0.18 g feed g −1 fish was found for T4 culture system.On the other hand, the lowest values of the fish growth rate, specific growth rate and feed efficiency ratio were 1.22 ± 0.23 g day −1 , 1.40 ± 0.11% day −1 and 0.86 ± 0.04 g fish g −1 feed, respectively, were found for T4 culture system, while, the highest value of the feed conversion ratio was 1.46 ± 0.16 g feed g −1 fish was found for T1 culture system.Better fish growth performance in T3 may be as a result of better water quality parameters.These results were in agreement with 32,33 .
From statistical analysis of the experimental data, there were no significant different between T1, T3 and T5 systems on the fish growth rate, specific growth rate, feed conversion ratio and feed efficiency ratio, meanwhile, there were significant differences among T4 system and T1, T3 and T5 systems on the fish growth rate, specific growth rate, feed conversion ratio and feed efficiency ratio.

Production costs
Table 5 shows the fixed, variable and total costs of tilapia fish and lettuce plant for different culture systems (T1: Aquaculture (control), T3: Aquaponics without nutrients addition, T4: Aquaponics with supplementary nutrients in water (EC is 800 ppm) and T5: Aquaponics with supplementary nutrients spray on plants) at the end growing period.It could be seen that the fixed costs were 3550, 2590, 4340, 4340 and 4340 LE for T1, T2, T3, T4 and T5 culture systems, respectively.The variable costs were 516, 230.5, 536.5, 544.4 and 545.4 LE for T1, T2, T3, T4 and T5 culture systems, respectively.The total production costs were 4066, 2820.5, 4876.5, 4884.4 and 4885.4LE for T1, T2, T3, T4 and T5 culture systems, respectively.From statistical analysis of the experimental data, there were no significant different between T3, T4 and T5 systems on the total production costs.

Conclusions
From this study, it is concluded that the highest values of N, P, k, Ca and Mg consumption rate were found with hydroponic standard requirement (T2) and 1.5 L h −1 plant −1 of flow rate, while, the lowest values of N, P, k, Ca and Mg consumption rate were found with aquaponics without nutrients additions (T3) and 2.0 L h −1 plant −1 of flow rate.The root length for lettuce plants grown in T2 system was taller than those grown in different culture system (T3, T4 and T5).The fresh and dry of shoot and root of lettuce plants grown in T2 system were better than those of different culture system (T3, T4 and T5).The highest value of individual fish weight was found for T3 culture system, while, the lowest value of individual fish weight was found for T4 culture system.The highest values of the fish growth rate, specific growth rate and feed efficiency ratio were found for T3 culture system, while, the highest value of the feed conversion ratio was found for T4 culture system.The production costs ranged from 2820.5 to 4885.4 LE ($ = 30.92LE) for all culture systems.Finally, to integrate between fish and plant, you must adjust and monitor the nutritional elements requirements and the flow rate to give both fish and plants their optimum requirements to get higher yield of both of them.Further studies are recommended to adjust the environmental factors the proposed system.FCR, g feed g −1 fish 1.46 ± 0.16 a 1.48 ± 0.17 a 1.58 ± 0.18 b 1.49 ± 0.16 a FER, g fish g −1 feed 0.98 ± 0.07 b 0.96 ± 0.05 b 0.86 ± 0.04 a 0.95 ± 0.06 b

Figure 2 .
Figure 2. The root length of Lettuce plants grown in different culture systems.

Figure 3 .
Figure 3. Fresh and dry weight of shoot of lettuce plants, (a) Fresh weight, and (b) Dry weight.

Figure 4 .
Figure 4. Fresh and dry weight of root of lettuce plants, (a) Fresh weight, and (b) Dry weight.

Figure 5 .
Figure 5. Individual fish weight for different culture systems.

Table 5 .
The total production costs of tilapia fish and lettuce plant for different culture systems.Means on the same row with different superscripts are significantly different (p < 0.05).